The genus Borrelia is included in the order Spirochaetales ("spirochetes") and family Spirochaetaceae. Borrelia species are associated with arthropod hosts and often have limited geographical ranges. Lyme borreliosis, a systemic illness with a wide spectrum of clinical symptoms, was named for Lyme, Conn., where the disease was recognized and studied in 1975.
The illness usually develops 3 to 30 days following the bite of an ixodid tick which transmits Borrelia burgdorferi to humans and animals. The disease in humans often begins with a primary skin lesion called erythema migrans (EM) which may be followed by cardiac, neurologic, or arthritic symptoms. The primary clinical sign in dogs and horses is lameness.
Antigenic proteins can be isolated from B. burgdorferi by immunoprecipitation, extraction from SDS polyacrylamide gels, or by molecular cloning and expression. The first two methods require large numbers of organisms, creating a logistical problem with B. burgdorferi, which has a generation time of 8 to 24 hours at 32.degree. C. and reaches a maximum cell density of 10.sup.7 to 10.sup.8 cells/ml.
Molecular cloning of protein antigens in a host such as Escherichia coli can result in their production in large amounts without contamination by other spirochete antigens. Using E. coli as a host for molecular cloning also avoids association of B. burgdorferi antigens with rabbit serum since the E. coli can be grown in Luria broth (LB) containing no rabbit serum. This is critical in the development of vaccines since spirochetal antigens may adsorb rabbit serum, a component of the medium used to propagate B. burgdorferi, potentially resulting in anaphylactic shock when administered to animals including humans.
Two major surface proteins with molecular weights of 31 kilodalton (-kD) and 34-kD (OspA and OspB, respectively) have been cloned, sequenced, and characterized. See, S. Bergstrom et al., Mol. Microbiol., 4, 479 (1989). Those two proteins are under the control of a single promoter and are found on a linear plasmid. See, T. R. Howe et al., Infect. Immunol., 54, 207 (1985).
The 41-kD protein, a flagellin component, has been cloned, sequenced and found to be recognized early in the immune response by A. G. Barbour et al., J. Clin. Invest., 72, 504 (1983) and R. Wallech et al., Infect. Immun., 58, 1711 (1990). Unfortunately, human antibodies specific to this flagellin component cross react with other species of Borrelia, thus reducing the specificity of potential serological assays for the diagnosis of Lyme disease which use the flagellin protein as the "capture antigen". C. Collins et al., Infect. Immunol., 59, 519 (1991).
A fourth immunodominant protein also has been cloned by K. Hansen et al., Infect. Immunol., 56, 2047 (1988). Antibodies to this 60-kD recombinant protein also cross reacts with a large variety of microorganisms including Pseudomonas and Legionella, therefore this antigen is not useful for diagnosis of Lyme borreliosis.
Recently, W. J. Simpson et al., J. Clin. Microbiol., reported cloning a 6.3 Kb EcoR1 chromosomal fragment of B. burgdorferi DNA, which encoded two proteins of 28-kD and 39-kD. These two antigens were reported to be immunologically distinct from OspA, OspB and the 41-kD flagellin protein.
Progress towards prevention and treatment of Lyme borreliosis in humans and domestic animals has been aided by the development of laboratory animal models exhibiting signs of Lyme borreliosis. Rabbits and guinea pigs develop skin lesions resembling human EM lesions but no other signs of disease. Hamsters develop arthritis when inoculated in the paw and are irradiated. Infant and weanling laboratory rats develop a persistent multisystemic infection, polyarthritis, and carditis after intraperitoneal (i.p.) inoculation of B. burgdorferi. Mice (C3H/He) develop spirochetemia, carditis, and polyarthritis after interperitoneal (i.p.) inoculation of B. burgdorferi. The last two animal models closely mimic human Lyme borreliosis.
With the development of laboratory animal models which have signs of Lyme borreliosis, research is in progress to determine if they can be protected from experimental B. burgdorferi infection and/or clinical manifestations of disease. The ability to protect laboratory animals from B. burgdorferi infection was first demonstrated by Johnson et al., Infect. Immunol., 54, 897 (1986) who showed that hamsters were protected from infection by immunization with formalin treated B. burgdorferi spirochetes. The hamsters were also protected by the administration of sera obtained from rabbits immunized with the spirochetes. (R. C. Johnson et al., Infect. Immunol., 53, 713 (1986)). At thirty days postvaccination with spirochetes, 86-100% protection against infection was exhibited by hamsters receiving 50 and 100 mg (dry weight) of this vaccine. However, resistance to infection decreased to 25% and 40% for the 100 mg and 50 mg vaccine doses, respectively, at 90 days post-vaccination.
Protection from infection and induction of Lyme arthritis also was shown in irradiated hamsters by the injection of B. burgdorferi and immune serum in the hind paw by J. L. Schmidtz et al., Infect. Immunol., 58, 144 (1990). Recently, Fikrig et al., in Science, 250, 553 (1990), reported that C3H/He laboratory mice were protected from infection and induction of Lyme arthritis by active immunization with the purified recombinant OspA protein.
Vaccines based on recombinant polypeptides have a number of potential advantages over vaccines based on the "killed" or inactivated parent organisms, including lack of infectivity and side effects, reproducibility and high antigenicity. Therefore a need exists for recombinant vaccines which are effective to protect mammals against B. burgdorferi infection (Lyme disease) for prolonged periods of time, while exhibiting minimal host toxicity.